Cytochromes P450 termed CYP3A4 and CYP3A5 are relatively abundant in human intestinal epithelial cells. During the prior funding interval, we have shown that CYP3A4 is inducible in human small bowel by rifampin treatment, and this appears to account in large part for the clinically important drug interaction between rifampin and the CYP3A4 substrate, cyclosporine A. We have also shown that there is >10 fold interindividual variation in the intestinal expression of both CYP3A4 and CYP3A5 in individuals not receiving known enzyme inducers. This heterogeneity may contribute to inter individual differences in oral clearance of at least some CYP3A substrates, and may also represent a previously unrecognized risk factor for some environmental diseases. The studies proposed in Specific Aims l, 2, and 3 will characterize the nongenetic factors (medications and diet) that may largely account for this variability. We will first identify potential inducers of CYP3A4 and CYP3A5 mRNA and protein in human small bowel and colon, utilizing an intestinal explant culture model and techniques (including quantitative polymerase chain reaction) developed during the prior funding period. In addition, we will test the hypothesis that substances routinely present in food are inducers of CYP3A4 and CYP3A5 in intestine by placing healthy subjects on diets containing cruciferous vegetables (indoles), charcoal broiled beef (combustion by- products) or grapefruit juice (flavonoids). Expression of CYP3A4 and CYP3A5 mRNA and protein will be determined in biopsies endoscopically obtained from small and large intestine at study entry, after a four day interval on an """"""""inducer free"""""""" diet, and again after 7 days on the test diet. We will also test the hypothesis that interindividual differences in responsiveness to CYP3A inducers is common and inducer class specific. This will be accomplished by performing endoscopic biopsies on healthy subjects before and after short term treatment with compounds identified (in the initial explant studies) to be CYP3A4 orCYP3A5 inducers. Responsiveness to inducers in vivo will be confirmed by induction studies performed in vitro in cultured intestinal explants obtained from each subject. Finally, we will directly assess the influence of interindividual variation in intestinal expression of CYP3A4 and/or CYP3A5 on """"""""first pass"""""""" metabolism by determining the oral kinetics of 3 important CYP3A substrates (cyclosporine A, midazolam, and 17 alpha ethinyl estradiol) in healthy volunteers who have undergone small bowel biopsy (Specific Aim 4). This proposal capitalizes on our laboratory's close association with The General Clinical Research Center, which is an ideal environment for the proposed studies. The data obtained should provide insight into the basis for interindividual differences in expression of CYP3A4 and CYP3A5 in the intestine, and should directly confirm the influence that this heterogeneity may have on the oral bioavailability of CYP3A substrates.
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